Application Of Quantitative PCR And Imaging Flow Cytobot In The Study Of Alexandrium Blooms

Harmful algal bloom(HAB) is a common ecological disaster. Some HAB species are toxigenic and paralytic shellfish toxins(PSTs) are the most widespread phycotoxin with severe impacts. The most important PST source in seawater is toxic species in Alexandrium genus(dinoflagellate). Therefore, the species-specific detection for toxic Alexandrium species and in-situ observation of Alexandrium blooms are significant issues in studies of harmfu algal blooms.Alexandrium tamarense species complex is a group of important HAB causative species in China. In this study, species-specific and PST-related gene detecting assays were applied into toxic A. tamarense species complex detection along Chinese coastal waters for the first time. In addition, the in-situ study of Alexandrium bloom dynamics monitoring using Imaging FlowC ytobot(IFCB) was conducted in US under the support of Chinese Scholarship Council. The summary is as follows:(1) Two TaqMan-based qPCR assays targeted at large subunit ribosomal DN A gene(LSU r DNA) of A. fundyense and A. pacificum belonging to A. tamarense species complex were established and tested for their specificity, sensitivity and accuracy. Those methods were applied to the detection of A. fundyense and A. pacificum in parallel with microscopy counting for Alexandrium population quantitation and high performance liquid chromatography(HPLC) for PST level measurements in Changjiang River estuary, Yellow Sea and the Bohai Sea. It is indicated that A. pacificum mainly presented in the sea area adjacent to the Changjiang River estuary in Spring 2011. Additiona lly, a small amount of A. pacificum was also found in the coastal waster of Q inhuangdao in the BS. A. fundyense mainly distributed northern to the sea area of 34 oN in the YS. This is the first report of the distribution pattern of A. fundyense and A. pacificum in the coastal waters of China. Meanwhile, the significant specificity and sensitivity of qPCR-based assays were exhibited which will be great potential in Alexandrium ecological study.(2) A SYBR Green qPCR assay targeted at the unique gene sxt A4 in saxitoxin biosynthesis was established and applied to monitor PST-producing algae in the YS in Spring 2012 and in sea area adjacent to the Changjiang River estuary in Spring 2013. In addition, TaqMan-based qPCR assays were employed to analyze toxic A. tamarense species complex and HPLC was used to analyze PSTs in parallel. It was found that the distribution pattern of sxt A gene coincided with those of A. fundyense/A. pacificum and PSTs, and sxt A-based qPCR results correlated well with abundances of toxic A. tamarense species complex derived from TaqMan-based qPCR assays(r2=0.734 in YS and 0.519 in Changjiang River estuary respectively). These suggest that toxic A. tamarense species complex were the major PST producers during the sampling season, and sxt A-based qPCR is a promising method to monitor PST-producing algae along Chinese coastal waters. The correlation between PST levels and sxt A-based qPCR results was less significant(r2=0.305 in YS), implying that sxt A-based qPCR is not enough to reflect the toxicity and potential impacts of PST-producing blooms. Hence, the combination of sxt A-based qPCR assay with chemical means could offer an even stronger approach in early-warning of toxic algal blooms.IFCB was applied into the study of toxic A. fundyense bloom dynamics in Salt Pond of Cape Cod, US. Two versions of classifier were built and compared for the automated quantitation of A. fundyense. The results showed that the 64 sub-classes version was superior to the 80 sub-classes version. There was significa nt relationship between automated classification results of 64 sub-classes classifier and corrected results(r2 =0.958). During the deployment, IFCB recorded the entire A. fundyense bloom process. In addition, it was the first time that Heterocapsa triquetra also bloomed during A. fundyense bloom and the cell abundance could reach 107 cells L-1. It showed that IFCB is an important harmful algal bloom detecting technology with great potential to improve the understanding of bloom mechanisms.In general, this study employed species-specific and sxt A-based qPCR assays into the detection of toxic Alexandrium in the BS, YS and East China Sea. It clarified the distribution pattern of two toxic Alexandrium species and indicated the source of PST in the investigated sea areas. It also verified the great value of molecular biological technology in HAB studies. In addition, the in-situ study of Alexandrium bloom using IFCB supported by China Scholarship Council indicated the sexual recombination phase and the role of the parasite Amoebophrya in the termination of the bloom. Those results improved the understanding of toxic Alexandrium bloom along Chinese coastal waters and provide important methodological basis for Alexandrium study.